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Endocrinology, doi:10.1210/en.2008-0547
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Endocrinology Vol. 149, No. 11 5568-5576
Copyright © 2008 by The Endocrine Society

Up-Regulation of 3'5'-Cyclic Guanosine Monophosphate-Specific Phosphodiesterase in the Porcine Cumulus-Oocyte Complex Affects Steroidogenesis during in Vitro Maturation

Maxime Sasseville, Nancy Côté, Marie-Claude Gagnon and François J. Richard

Centre de Recherche en Biologie de la Reproduction, Département des Sciences Animales, Université Laval, Québec, Canada G1K 7P4

Address all correspondence and requests for reprints to: François J. Richard, Ph.D., Centre de Recherche en Biologie de la Reproduction, Département de Sciences Animales, Université Laval, Québec, Canada G1V 0A6. E-mail: francois.richard{at}fsaa.ulaval.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The 3'5'-cyclic GMP (cGMP) pathway is known to influence ovarian functions, including steroidogenesis, ovulation, and granulosa cell proliferation. We show here that cGMP-phosphodiesterase (PDE) activity increased in a gonadotropin-dependent manner more than 3-fold in the cumulus-oocyte complex (COC) after 24 h in vitro maturation (IVM) and up to 5-fold after 48 h. Further characterization of this increase demonstrated that the activity was located primarily in cumulus cells, and was sensitive to sildenafil and zaprinast, two inhibitors specific to both type 5 and 6 PDEs. RT-PCR experiments showed that the mRNAs for cGMP-degrading PDEs 5A and 6C are present in the COC before and after 30 h IVM. Western blotting confirmed the presence of PDE 5A in the COC. Western blotting of PDE 6C revealed a significant up-regulation in the COC during IVM. Isolation and analysis of detergent-resistant membranes suggested that PDE 6C protein, along with half of the total sildenafil-sensitive cGMP-degradation activity, is associated with detergent-resistant membrane in the COC after 30 h IVM. Treatment of porcine COC with sildenafil during IVM caused a significant decrease in gonadotropin-stimulated progesterone secretion. Together, these results constitute the first report exploring the contribution of cGMP-PDE activity in mammalian COC, supporting a functional clustering of the enzyme, and providing the first evidence of its role in steroidogenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE CYCLIC NUCLEOTIDE second messengers cAMP and 3'5'-cyclic GMP (cGMP) are important for signal transduction, and they mediate a wide range of cellular processes (1). They are synthesized by adenylyl cyclase and guanylyl cyclase, respectively, and their degradation is catalyzed by members of the phosphodiesterase (PDE) protein family (2). The role of cyclic nucleotides in ovarian physiology has been explored for the last 40 yr.

Cyclic GMP can be generated through the nitric oxide synthase (NOS)/nitric oxide (NO)/soluble guanylyl cyclase pathway or by the natriuretic peptide/membrane-bound guanylyl cyclase pathway. Members of these two pathways are expressed in the mammalian ovary. NOS2 and NOS3 (previously known as inducible and endothelial NOS) are expressed in the pig ovary (3). Two isoforms of soluble guanylyl cyclases are expressed in rat ovaries (4). Numerous studies have described the effects of the NO/cGMP pathway on various physiological functions of granulosa cells (5). NO releasing agents and cGMP analogs have perturbed steroid secretion in rat (6), pig (7), and human granulosa cells (8), as well as modified gene expression (6, 8, 9). NO releasing agents have also inhibited apoptosis in rat granulosa cells (10). Mice oocyte nuclear maturation is impaired by genetic disruption of the NOS3 gene (11). Female mice lacking the gene for natriuretic peptide receptor B had smaller ovaries and no follicles larger than the secondary stage, and they were infertile (12). Furthermore, atrial natriuretic peptides (ANPs) have affected oocyte maturation in rats and pigs (13, 14).

Of the 11 PDE families known, eight hydrolyze cGMP with different efficiencies. These eight families are separated into two groups: the dual substrate-specificity group and the cGMP-specific group. The first group is composed of PDE1, PDE2 PDE3, PDE10, and PDE11. From these, only PDE1 and PDE3 have been expressed in the ovary (15). The cGMP-specific PDE group is composed of PDE5, PDE6, and PDE9. Only PDE9A mRNA is expressed in the ovary (16).

Although a remarkably large body of literature describes the vast array of effects of cGMP-generating pathways on the functions of the ovarian follicle, no study has yet explored the expression and regulation of cGMP-PDEs in the ovarian follicle of any species. The purpose of the present study was to assess the expression, activity, and role of cGMP-PDEs in porcine cumulus-oocyte complexes (COCs).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Ovary collection
Prepubertal pig ovaries were collected according to the method used by Laforest et al. (17). In short, they were recovered from a local slaughterhouse and maintained at 34 C in saline (0.9% NaCl) containing the following antibiotics and antimycotics: 100,000 IU/liter penicillin G, 100 mg/liter streptomycin, and 250 mg/liter amphotericin B. Upon arrival from the slaughterhouse, they were rinsed in saline with antibiotics and antimycotics at 34 C.

Chemicals
Unless otherwise noted, all chemicals were bought from Sigma Chemical Co. (St. Louis, MO). Zaprinast was bought from Calbiochem (San Diego, CA). Sildenafil was a generous gift from Sharron H. Francis (Vanderbilt University Medical Center, Nashville, TN), and it was purified from tablets following a previously published protocol (18). All pharmacological inhibitors were stored in aliquots at –80 C until use.

COC culture conditions
COCs were cultured as previously described (19, 20). They were collected in centrifuged follicular fluid and selected on the basis of a homogeneous oocyte cytoplasm and a compact cumulus cell mass. Immediately before in vitro culture, they were washed three times in HEPES-buffered Tyrode medium containing 0.01% (wt/vol) polyvinyl alcohol (PVA) (PVA-HEPES) (21). COCs (15 to50) were cultured in 500 µl in vitro maturation (IVM) medium in Nunclon {Delta}4 four-well plates. IVM medium is a BSA-free NSCU23-based medium supplemented with 25 µM β-mercaptoethanol (Bio-Rad Laboratories, Inc., Hercules, CA), 0.1 mg/ml cysteine, 10% (vol/vol) porcine follicular fluid, and gonadotropin supplements at final concentrations of 2.5 IU/ml human chorionic gonadotropin (Intervet, Whitby, Ontario, Canada) and 2.5 IU/ml equine chorionic gonadotropin (Intervet). Before use the follicular fluid and gonadotropin supplements were sterilized by passage through a 0.22-µm filter.

Cyclic GMP-PDE assay
Cumulus cells and denuded oocytes samples were generated by vigorously pipetting COC with a P200 and selecting denuded oocytes with a glass pipette. Before freezing, COC, cumulus cells, and denuded oocytes were washed twice in PVA-HEPES media. For cGMP-PDE activity measurement, samples were suspended in hypotonic buffer, which contained 20 mM Tris-HCl (pH 7.4), 1 mM EDTA, 0.2 mM EGTA, 50 mM sodium fluoride, 50 mM benzamidine, 10 mM sodium pyrophosphate, 4 µg/ml aprotinin, 0.7 µg/ml pepstatin, 10 µg/ml soybean trypsin inhibitor, 0.5 µg/ml leupeptin, and 2 mM phenylmethylsulfonyl fluoride. The suspension was homogenized by seven to nine freeze/thaw cycles and vortexing (20). In all experiments, 0.5% (vol/vol) Triton X-100 was added to the hypotonic buffer as a detergent. The homogenate was centrifuged for 20 min at 13,000 x g to obtain the supernatant. The enzymatic activity was assessed using 1 µM cGMP as substrate as described in previous studies (22, 23). Samples equivalent to 10 COCs per assay tube were used throughout the study. The sample volume was adjusted to 150 µl with 3[N-morpholino]propanesulfonic acid (MOPS)-BSA solution [40 mM MOPS (pH 7), containing 1 mg/ml fraction V BSA]. The reaction was initiated by adding 50 µl incubation mix [40 mM MOPS (pH 7), 0.8 mM EGTA, 5 mM magnesium acetate, 1 µM cold cGMP, and [8-3H]cGMP (GE Healthcare, Baie d’Urfé, Quebec, Canada) (1 x 105 cpm/tube; 5–25 Ci/mmol)] and incubating at 34 C for 20 min. Reactions were terminated by incubation in boiling water for 1 min. Cyclic GMP that was hydrolyzed by PDE into guanosine 5'-monophosphate was then turned into guanosine by adding excess Crotalus atrox venom (5'-nucleotidase) (50 µl, 1 mg/ml) and incubating 15 min at 34 C. Guanosine was separated from intact cGMP by anion-exchange chromatography. The amount of tritiated guanosine was quantified by liquid scintillation. Each measurement was done in triplicate within a single assay to account for intraassay variation. The experiment was performed independently at least three times. The final concentrations of the inhibitors used were 1 mM 3-isobutyl-1-methylxanthine (IBMX), 100 µM zaprinast, and 10 µM sildenafil.

RNA extraction and RT-PCR
COCs before and after 30 h IVM were rinsed twice in PVA-HEPES media and frozen in liquid nitrogen until processed. RNA extractions were performed with the Absolutely RNA Microprep Kit from Stratagene (La Jolla, CA) according to the manufacturer’s protocol. RNA samples were eluted in 15 µl, followed by reverse transcription using the OmniScript RT Kit from QIAGEN, Inc. (Valencia, CA), and poly (deoxythymidine) from Ambion, Inc. (Austin, TX). The primer pairs were designed according to bovine β-actin (ACTB) (AY141970.1), pig PDE5A (AY266366.1), bovine PDE6A (NM_001001526), bovine PDE6B (NM_174418), and bovine PDE6C (NM_174419). Primer sequences and expected PCR product sizes are shown in Table 1Go. Primers were purchased from Integrated DNA Technologies (Skokie, IL). PCRs were performed in a 50-µl reaction volume using FastStart Taq polymerase from Roche Diagnostics (Laval, Quebec, Canada). Cycling conditions for all amplification reactions were as follows: 2 min at 95 C; 32 cycles of 1 min at 95 C, 1 min at 58 C, and 1 min at 72 C; and 10 min at 72 C. Positive controls were performed in parallel with 1 fg purified and sequenced PCR products amplified using porcine testis cDNA as the template. Additional amplifications were performed using ACTB primers on an equivalent amount of RNA to detect effects due to residual contamination with genomic DNA. Amplifications were visualized by 1% agarose gel electrophoresis and ethidium bromide staining. PCR products were purified using a gel purification kit (QIAGEN), and they were sequenced to confirm their identity.


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  		TABLE 1. Primers used to perform PCR amplifications

 
Western blotting
Samples were collected and processed in hypotonic buffer as described previously in the cGMP-PDE assay section. Protein samples were loaded on a 12% SDS-PAGE. Proteins were transferred to a Hybond-P membrane (GE Healthcare) using a Mini Protean 3 Cell apparatus (Bio-Rad Laboratories Ltd., Mississauga, Ontario, Canada). Membranes were blocked with Tris-buffered saline containing 0.1% (vol/vol) Tween 20 and 5% (wt/vol) nonfat dry milk. Membranes were blotted with the primary antibody anti-PDE5A (diluted 1:500; FabGennix Inc., International, Frisco, TX) at room temperature for 2 h, after which the membranes were exposed to goat antirabbit peroxidase-conjugated secondary antibody (diluted 1:10,000; Jackson ImmunoResearch Laboratories Inc., West Grove, PA). PDE6C detection was performed using a PDE6C antibody (diluted 1:1,000; Affinity BioReagents, Inc., Golden, CO), followed by a detection with a goat antirabbit peroxidase-conjugated secondary antibody (diluted 1:20,000; Jackson Immunoresearch Laboratories). For {alpha}-tubulin detection, a primary anti-{alpha}-tubulin antibody (Upstate, Charlottesville, VA) was diluted 1:50,000 in Tris-buffered saline with 0.1% Tween 20. Membranes were blotted with a horseradish peroxidase-conjugated goat antimouse (diluted 1:20,000; Upstate). Binding was detected using the ECL Plus kit (GE Healthcare) and exposed on autoradiographic films (GE Healthcare). Densitometric analysis of images was performed using ImageJ version 1.37 software (National Institutes of Health, Bethesda, MD).

Detergent-resistant membrane (DRM) isolation by sucrose density gradient
The lipid content of rafts contributes to their hydrophobic nature and leads to two inherent biochemical properties: insolubility at 4 C in Triton X-100, and light buoyant density after centrifugation in a sucrose density gradient. These properties are used to isolate DRM fractions as biochemical correlates of lipid rafts (24). DRMs were isolated as previously reported, with minor modifications (25). Briefly, 1200–1400 COCs were subjected to eight freeze/thaw cycles in ice-cold 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 5 mM EDTA. The cells were further lysed in 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 5 mM EDTA containing 1% (vol/vol) Triton X-100 for 30 min on ice. The lysates were mixed with an equal volume of 85% (wt/vol) sucrose, and overlaid with 2.4 ml 35% (wt/vol) sucrose and 1 ml 5% (wt/vol) sucrose. Centrifugation was performed at 39,000 x g in a Beckman SW60Ti rotor at 4 C for 18 h using an OptimaXL-80K Beckman-Coulter centrifuge (Beckman Coulter, Inc., Fullerton, CA). A total of 11 fractions (380 µl) were collected starting from the top of the tube. Fractions 2–5 were considered to contain DRM, fractions 6–8 were the intermediate fractions, and fractions 9–11 were the detergent-soluble fractions. Protein quantification in each sample was assessed by the bicinchoninic acid protein assay (Pierce, Rockford, IL). Each DRM isolation experiment was validated by determining enrichment of monosialoganglioside GM1 in the DRM fractions (data not shown). Each fraction (10 µl) was dotted onto Hybond-P membranes using a dot spot apparatus (Invitrogen Corp., Carlsbad, CA). The membranes were incubated with a 1:3000 dilution of horseradish peroxidase-conjugated cholera toxin B subunit and detected using an ECL Plus kit as described previously. Cholera toxin B subunit binds specifically to GM1, a known marker of lipid rafts (26).

Progesterone quantification
To measure progesterone secretion, COCs were cultured in 96-well plates (10 COC/125 µl) and incubated for 48 h in IVM medium. For each experimental condition, three independent culture experiments were performed, and media were centrifuged and cryopreserved. Progesterone was quantified in each media sample in triplicate using a commercial progesterone enzyme immunoassay (ALPCO Diagnostics, Salem, NH) following the manufacturer’s instructions. The sensitivity of the kit for progesterone was 0.1 ng/ml according to the manufacturer.

Statistical analysis
Statistical analyses were conducted using Prism 5.00 GraphPad for Windows (GraphPad Software Inc., San Diego, CA; www.graphpad. com). Statistical significance was assessed by ANOVA, followed by either Dunnett’s (Figs. 1Go and 2Go) or Bonferroni’s (Figs. 4Go and 6Go) multiple-comparison post hoc tests to identify individual differences between means. Probabilities of P < 0.05 were considered statistically significant. All values are presented with their corresponding SEM.


Figure 1
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  		FIG. 1. Profiling cGMP degradation in the COC during IVM. A, cGMP-PDE activity measurements in the COC every 6–9 h IVM. B, Sensitivity of cGMP-PDE activity in the COC to various PDE inhibitors after 24 h IVM (IBMX, nonspecific inhibitor; zaprinast and sildenafil, PDE5/PDE6-specific inhibitors). C, Effect of gonadotropins on cGMP-PDE activity after 30 h IVM. Cyclic GMP PDE activity was assessed in COCs before and after 30 h IVM with or without gonadotropins. The averages of three experiments are shown.

 

Figure 2
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  		FIG. 2. Cell type-specific enrichment of cGMP-PDE activity in the COC after 24 h IVM. Cell extracts were prepared from intact COCs (A), COC-cultured cumulus cells (B), or COC-cultured denuded oocytes (C). They were then assessed for cGMP-PDE activity. The averages of three experiments are shown. The asterisks represent significant statistical difference (P < 0.05) with the measurement without inhibitors as determined by ANOVA analysis followed by Dunnett’s post hoc test.

 

Figure 4
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  		FIG. 4. PDE5A and PDE6C immunodetection in COCs during IVM. A, The effect of IVM on PDE5A immunodetection in the COC. Lane 1, 25 µg mouse lung protein extract. Lane 2, 50 µg pig penile cavernous tissue protein extract. Lane 3, 50 COCs before IVM. Lane 4, 50 COCs after 30 h IVM. B, The same samples as in A were immunoblotted for {alpha}-tubulin. C, The effect of IVM on PDE6C immunodetection in COCs. Lane 1, 25 µg pig retina tissue protein extract. Lane 2, 50 COCs before IVM. Lane 3, 50 COCs after 30 h of IVM. D. The same samples as in C were immunoblotted for {alpha}-tubulin. For parts A–D, the upper panel is a representative experiment of samples immunoblotted for PDE5A (A) or PDE6C (C). The lower panel represents the same samples immunoblotted for {alpha}-tubulin (B and D). E, The bar graph presents the mean ratio of PDE to {alpha}-tubulin protein levels in three experiments. The molecular masses (in kDa) of protein markers are shown on the left. A–D were generated by grouping sections of the same gel.

 

Figure 6
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  		FIG. 6. Effect of PDE5/6 inhibition using sildenafil on progesterone secretion of untreated and gonadotropin-treated COCs after 48 h IVM. Each bar presents the mean of three separate experiments.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cyclic GMP-PDE activity in COCs during IVM
COCs were cultured in IVM medium, and cGMP degradation activity was assessed at regular intervals up to 48 h later. Our results show that cGMP-PDE activity increases 3.1-, 4.7-, 5.5-, and 5.5-fold after 24, 30, 39, and 48 h IVM, respectively (P < 0.05) (Fig. 1AGo). Further analysis of this cGMP-PDE increase revealed that the activity measured after 24 h IVM in the COC was significantly inhibited by IBMX (1 mM, nonspecific inhibitor), zaprinast (100 µM, PDE5/6-specific), and sildenafil (10 µM, PDE5/6-specific) (P < 0.05) (Fig. 1BGo), supporting the idea that PDE5 and PDE6 families contribute to the increased activity (27). To verify the gonadotropin dependence of the cGMP-PDE activity increase, cGMP degradation was measured in COCs after 30 h IVM with or without gonadotropins. Our results show a significant increase in cGMP-PDE activity only in gonadotropin-treated COCs (P < 0.05) (Fig. 1CGo). These results suggest that gonadotropins stimulate type 5 and/or type 6 PDE activity in COCs during IVM.

To localize the compartment in which the increase in cGMP degradation occurs, cGMP-PDE activity was measured separately in the oocyte (Fig. 2CGo) and the surrounding cumulus cells (Fig. 2BGo) after 24 h IVM. In these experiments 70% of cGMP-PDE activity was present in the cumulus cells, whereas only 30% of the activity was measured in the oocyte (Fig. 2Go). Moreover, the cGMP-PDE activity present in the cumulus cells was significantly inhibited by the nonspecific inhibitor IBMX and the PDE5/6-specific inhibitor zaprinast (P < 0.05) (Fig. 2BGo), further supporting the presence of PDE5 and/or PDE6 in cumulus cells.

Identification of sildenafil- and zaprinast-sensitive PDEs
There is a single type 5 PDE gene and three type 6 PDE genes currently known: PDE5A, PDE6A ({alpha}-subunit), PDE6B (β-subunit), and PDE6C ({alpha}' subunit) (28). mRNA expression patterns in porcine COCs before and after 24 h IVM were investigated using RT-PCR analysis. Figure 3Go shows that bands at the expected size are detected both in COC cDNA after 0 h (lane 1) and 24 h (lane 2) IVM for ACTB (Fig. 3AGo). PDE5A (Fig. 3BGo) and PDE6C (Fig. 3EGo) transcripts are also detected in COCs after 0 and 24 h IVM, although there seemed to be less expression after 24 h. Our experiments failed to detect PDE6A (Fig. 3CGo) and PDE6B (Fig. 3DGo) in porcine COCs before and after 24 h IVM. Amplification products were sequenced and found to share homology of 92.8, 92.3, 95.3, and 94.3%, respectively, to pig PDE5A and bovine PDE6A, PDE6B, and PDE6C, as reported in GenBank (data not shown). The higher molecular mass PCR product in the PDE5A amplification could not be sequenced using our PCR primers and was, therefore, ignored. These results suggest that PDE5A and PDE6C are present in COCs during IVM.


Figure 3
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  		FIG. 3. RT-PCR detection of ACTB (A), PDE5A (B), PDE6A (C), PDE6B (D), and PDE6C (E). For every gel, the predicted size (in base pairs) based molecular mass markers are indicated on the left (M). RT-PCR amplifications were conducted on three different biological samples of COCs using RNA isolated from before IVM (lane 1) or after 24 h IVM (lane 2). PCR amplifications were also conducted without template (lane 3) or on diluted PCR products already identified by sequencing (lane 4).

 
Based on these findings, PDE5A and PDE6C proteins present in COCs were investigated by Western blot analysis. One band was observed in positive controls, mouse lung, and in pig penile cavernous tissues, at the approximate 100-kDa size expected for PDE5A (lanes 1 and 2, Fig. 4AGo). PDE5A protein detection was observed in COCs before (lane 3, Fig. 4AGo) and after 30 h (lane 4, Fig. 4AGo) IVM. Densitometric analysis relative to {alpha}-tubulin levels (Fig. 4BGo) revealed no significant variations between the two time points (Fig. 4EGo). PDE6C immunodetection showed two bands of approximately 60 and 85 kDa in an extract of pig retina proteins (lane 1, Fig. 4CGo). Similar bands were present in COCs (lane 2 and 3, Fig. 4CGo), and PDE6C quantification relative to the level of {alpha}-tubulin (Fig. 4DGo) showed a significant increase of PDE6C in COCs from 0–30 h IVM (P < 0.05) (Fig. 4EGo). Our investigations also have shown that this up-regulation could be obtained using recombinant human FSH and that no increase was observed after 30 h if no gonadotropins were added (supplemental Fig. 1, which is published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org), supporting the cGMP-PDE activity measurements (Fig. 1CGo). These data strongly support the presence of the transcript and the protein of PDE5A and PDE6C in the COC (Figs. 3Go and 4Go). They further demonstrate that the increase in cGMP-PDE activity is caused by PDE6C up-regulation.

To assess the intracellular localization of the cGMP-PDE activity, light fractions (containing DRMs), intermediate fractions, and heavy fractions (containing detergent-soluble material) from a sucrose density gradient of COC extracts after 30 h IVM were submitted to cGMP degradation analysis Western blotting for PDE5A and PDE6C. Cyclic GMP-PDE activity measurements showed that approximately 37% of cGMP degradation activity was localized in the DRM and 45% in detergent-soluble fractions (Fig. 5AGo), suggesting a clustering of cGMP-PDEs in the DRM. Moreover, sildenafil (10 µM) inhibited 84 and 68% of the activity measured in DRM and detergent-soluble fractions, respectively (Fig. 5AGo). It is worth noting that similar levels of sildenafil-sensitive cGMP-PDE activity were observed in DRM and detergent-soluble fractions, even though there was 5-fold less protein in the DRM (Fig. 5BGo), suggesting a preferential association of cGMP-PDE activity with the DRM. PDE5A was detected by Western blotting only in intermediate and detergent-soluble fractions (lanes 3 and 4, Fig. 5CGo). PDE6C was detected in DRM fractions (lane 2, Fig. 5DGo), intermediate fractions (lane 3, Fig. 5DGo), as well as detergent-soluble fractions (lane 4, Fig. 5DGo) extracted from COCs after 30 h IVM. This corroborates the results of the cGMP-PDE activity assay. The results obtained so far strongly suggest that PDE5A and PDE6C are both expressed and active in COCs during IVM, and that PDE6C is up-regulated by gonadotropins and clusters in DRM during IVM.


Figure 5
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  		FIG. 5. Cyclic GMP-PDE activity and PDE5A/PDE6C immunodetection in different cellular fractions of COCs during IVM. A, Cyclic GMP-PDE activity was measured in DRM fractions, intermediate (Inter) fractions, and detergent-soluble fractions, as well as in crude protein extract. The activity was assessed alone or in the presence of sildenafil. PDE activity is plotted as the mean percentage of activity present in each fraction out of the total activity in crude protein extract from two experiments. B, Protein content of each fraction was assessed by bicinchoninic acid protein assay. C, Distribution of PDE5A in the DRM fractions, intermediate fractions, and detergent-soluble fractions of COCs at 30 h IVM. Lane 1, 50 µg pig penile cavernous tissue protein extract. Lanes 2–4, Protein samples from COCs after 30 h IVM of DRM (lane 2), intermediate fractions (lane 3), and detergent-soluble fractions (lane 4). D, Distribution of PDE6C in COCs after 30 h IVM. Lane 1, 25 µg pig retina tissue protein extract. Lanes 2–4, Protein samples from COCs after 30 h IVM with the DRM fractions (lane 2), intermediate fractions (lane 3), and detergent-soluble fractions (lane 4). Blots are representative of two separate experiments.

 
The effect of PDE5/6 inhibition on progesterone secretion
To evaluate the potential role of sildenafil-sensitive cGMP-PDE in the steroidogenesis of cumulus cells, COCs were treated with sildenafil (10 µM) during IVM, and the amount of progesterone in the medium was quantified by enzyme immunoassay. Data suggest that inhibition of both type 5 and 6 PDEs results in a significant reduction in the amount of progesterone secreted by gonadotropin-stimulated COCs (P < 0.05), whereas it does not affect nonstimulated COC progesterone secretion (Fig. 6Go). This result indicates that inhibition of cGMP-PDE during IVM regulates progesterone secretion.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, the expression, activity, and role of cGMP-degrading PDEs were examined in COCs. The data are showing that: 1) a zaprinast- and sildenafil-sensitive cGMP-PDE activity is mainly measured in cumulus cells and significantly increased by gonadotropins during IVM; 2) PDE5A and PDE6A mRNA and protein are present in COCs; 3) PDE6C protein increases during IVM and functionally clusters in the DRM; and 4) sildenafil treatment of COCs during IVM decreases progesterone secretion, thus supporting a functional role of PDE6C in steroidogenesis during IVM.

PDE5A is expressed in a wide variety of tissues, namely aortic smooth muscle cells, heart, placenta, and skeletal muscle, and pancreas, brain, liver, lung, and penile corpus cavernous tissue (29, 30). PDE6A and PDE6B form a heterodimer that is found almost exclusively in retinal rods (31). However, PDE6C forms a homodimer shown to be present in the retinal cone (32). As reviewed previously (33), there are no reports of the catalytic subunits of PDE6 expression (PDE6A, PDE6B, and PDE6C) other than in retina, pineal gland, and retina-derived tumors. Our study is the first to characterize the presence of cGMP-PDEs in the COC of any species.

The gonadotropin-stimulated increase of cGMP-PDE in the COC is likely to reflect the establishment of a cGMP degradation mechanism in the luteinizing follicular somatic cells. The increase in cGMP degradation mechanism in follicular somatic cells after a post-gonadotropin surge is further supported by several studies. Equine chorionic gonadotropin stimulated ANP and C-type natriuretic peptides (CNPs) in whole rat ovary (34). Equine chorionic gonadotropin also up-regulated CNPs in rat theca cells and natriuretic peptides receptor B in granulosa cells. This suggests possible paracrine communication involving cGMP signaling in the peri-ovulatory ovarian follicle (35). Up-regulation of cGMP-stimulated protein kinase 2 in mouse occurred in granulosa and cumulus cells in response to treatment with human chorionic gonadotropin (36). Concomitant with this up-regulation is the increased expression of several members of the cGMP synthesis pathway, namely natriuretic peptide receptor A, ANP, and CNP. Bovine corpus luteum cell membranes were shown to bind ANP and stimulate guanylyl cyclase activity, and to display significant cGMP-PDE activity (37, 38). Although none of these events has been studied closely in pigs, they strongly suggest that the cGMP signaling pathway is activated after gonadotropin stimulation in the follicular somatic cells. The rapid regulation of PDE6C in cumulus cells is in agreement with the regulation of this family in photoreceptor cells (33).

The effect of PDE5/6 inhibition on progesterone secretion suggests that cGMP degradation is affecting steroidogenesis during IVM. ANP has been studied widely for its effect on cGMP signaling and steroid secretion in the ovary. ANP stimulated cGMP accumulation and progesterone secretion in human granulosa-lutein cells (39). ANP also stimulated cGMP production and decreased rat ovarian steroidogenesis (34, 40). Another study showed that cGMP membrane-permeable analog decreased rat granulosa cell steroidogenesis in a dose-dependant manner (41). The precise mechanism by which PDE5/6 inhibition reduces steroidogenesis remains to be determined. NO was also shown to inhibit aromatase activity (42). Interestingly, analogs of cGMP affected steroidogenesis in pig mural granulosa cells in a biphasic manner (7). There are many cyclic GMP targets in the cell. Cyclic GMP can activate cGMP-stimulated protein kinase G, and alternatively it can increase cAMP levels by competing for PDE3 binding. Interestingly, pig cumulus cells up-regulate PDE3A in response to gonadotropins, which is unique to this species, as far as is known (43).

Previous studies suggested the presence of a type 5 and/or 6 PDE in the ovarian follicle and that it might play a role in oocyte nuclear maturation. It was shown that spontaneous and follicular fluid meiosis activating sterol-stimulated mouse oocyte nuclear maturation was inhibited by the PDE5/PDE6-specific inhibitor zaprinast (44). A study in pig COC has shown that zaprinast could increase ANP inhibition of oocyte first polar body extrusion, suggesting the presence of a PDE5/PDE6 enzyme in the COC (14). PDE3A, the principal PDE expressed in pig oocyte, has a similar affinity for cAMP and cGMP but hydrolyzes the former 10 times faster (28). It has been suggested that cGMP could serve as an inhibitor of oocyte meiotic resumption by preventing cAMP degradation in the oocyte by competing for PDE3A catalytic pocket (45). Cyclic GMP-PDE activity increase in the COC could then provide a mechanism by which cGMP would be depleted, thereby releasing cAMP inhibition of meiosis. However, pig COCs display only a weak cGMP-PDE activity before IVM, and that the increase occurs after 24 h IVM, where 85% of oocytes have undergone germinal vesicle breakdown in our in vitro system (20). Moreover, it was previously shown that porcine oocytes are irreversibly committed to resume meiosis 3 h before germinal vesicle breakdown. Therefore, the present increase in cGMP-PDE activity is unlikely to be part of the spontaneous meiotic resumption mechanism of porcine oocytes in vitro. However, it does not exclude the possibility that cGMP-PDE activity up-regulation might be a part of in vivo oocyte nuclear maturation.

The intracellular distribution of PDEs creates an additional level of complexity in cyclic nucleotide signaling. Although PDE5A is cytoplasmic, PDE6C is predicted as a membrane protein and is associated with DRMs in bovine retinal cells (46, 47). Our results show that PDE6C becomes located in DRM fractions (Fig. 5DGo), which would be consistent with the posttranslational isoprenylation observed in retinal cells (48). The presence of PDE6C in detergent-soluble fractions suggests either partial isoprenylation or the presence of the 17-kDa prenyl-binding protein (PrBP) (PrBP/{delta}) in the COC, which has been shown to solubilize PDE6C in retinal cones. PrBP/{delta} expression has never been studied in the ovarian follicle, but it may allow intracellular relocation of PDE6C.

The cGMP-PDE activity increase characterized here is based on cGMP-PDE measurements and immunodetection of PDE. These experiments cannot exclude an additional level of activity regulation such as phosphorylation. Wnt5a increased PDE6 activity 10-fold in mouse F9 teratocarcinoma cells expressing Frizzled2 in a p38 MAPK-dependent manner (49). Interestingly, gonadotrophins have up-regulated that pathway in mouse and pig cumulus cells (50, 51). Several studies have reported PDE regulation in the ovarian follicle. Rodent granulosa cells responded to gonadotropin stimulation by up-regulating a Ca2+/calmodulin-sensitive PDE and multiple splice variants of the PDE4D gene (52, 53). Human granulosa cells similarly up-regulated cAMP-PDE in response to gonadotropins (54, 55). We have recently reported that porcine cumulus cells up-regulate PDE3A in a cAMP/protein kinase A-dependent manner (43).

The present study has investigated cGMP degrading enzymes in COCs. Sildenafil-sensitive cGMP degradation was activated by gonadotropins in the cumulus cells during IVM. PDE5A and PDE6C mRNAs were expressed in the COC, and the level of PDE6C protein increased after 30 h IVM, and the protein clustered with the DRM. Furthermore, sildenafil treatment reduced gonadotropin-stimulated progesterone secretion of COCs during IVM. These results provide a new target for pharmaceutical modulation of cGMP signaling in the ovarian follicle, which may lead to a deeper understanding of the follicular terminal differentiation leading to ovulation.


   Acknowledgments
 
Sildenafil was kindly donated by Sharron H. Francis (Vanderbilt University Medical Center, Nashville, TN). We thank Isabelle Laflamme for professional and technical assistance. We also thank Richard Prince for collecting ovaries at the slaughterhouse and Dr. Zuzana Becotte Capova for providing porcine testis cDNA.


   Footnotes
 
M.S. is supported by a Natural Science and Engineering Research Council of Canada Ph.D. fellowship. This project was supported by the Natural Science and Engineering Research Council of Canada and by the Canadian Institutes for Health Research in the Program for Oocyte Health (to F.J.R.).

Present address for M.S.: Research Centre for Reproductive Health, School of Paediatrics and Reproductive Health, Discipline of Obstetrics and Gynaecology, Medical School, University of Adelaide, Adelaide 5005, Australia.

Disclosure Statement: The authors have nothing to declare.

First Published Online July 31, 2008

Abbreviations: ANP, Atrial natriuretic peptide; cGMP, 3'5'-cyclic GMP; CNP, C-type natriuretic peptide; COC, cumulus-oocyte complex; DRM, detergent-resistant membrane; IBMX, 3-isobutyl-1-methylxanthine; IVM, in vitro maturation; MOPS, 3[N-morpholino]propanesulfonic acid; NO, nitric oxide; NOS, nitric oxide synthase; PDE, phosphodiesterase; PrBP, prenyl-binding protein; PVA, polyvinyl alcohol.

Received April 16, 2008.

Accepted for publication July 22, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Conti M, Beavo J 2007 Biochemistry and physiology of cyclic nucleotide phosphodiesterases: essential components in cyclic nucleotide signaling. Annu Rev Biochem 76:481–511[CrossRef][Medline]
  2. Hardman JG, Robison GA, Sutherland EW 1971 Cyclic nucleotides. Annu Rev Physiol 33:311–336[CrossRef][Medline]
  3. Tao Y, Fu Z, Zhang M, Xia G, Yang J, Xie H 2004 Immunohistochemical localization of inducible and endothelial nitric oxide synthase in porcine ovaries and effects of NO on antrum formation and oocyte meiotic maturation. Mol Cell Endocrinol 222:93–103[CrossRef][Medline]
  4. Shi F, Stewart Jr RL, Perez E, Chen JY, LaPolt PS 2004 Cell-specific expression and regulation of soluble guanylyl cyclase {alpha} 1 and β 1 subunits in the rat ovary. Biol Reprod 70:1552–1561[Abstract/Free Full Text]
  5. LaPolt PS, Leung K, Ishimaru R, Tafoya MA, You-hsin Chen J 2003 Roles of cyclic GMP in modulating ovarian functions. Reprod Biomed Online 6:15–23[Medline]
  6. Chen YH, Tafoya M, Ngo A, LaPolt PS 2003 Effects of nitric oxide and cGMP on inhibin A and inhibin subunit mRNA levels from cultured rat granulosa cells. Fertil Steril 79(Suppl 1):687–693
  7. Sirotkin AV, Makarevich AV, Genieser HG, Kotwica J, Hetenyi L 2000 Effect of four cGMP analogues with different mechanisms of action on hormone release by porcine ovarian granulosa cells in vitro. Exp Clin Endocrinol Diabetes 108:214–219[CrossRef][Medline]
  8. Kagabu S, Kodama H, Fukuda J, Karube A, Murata M, Tanaka T 1999 Inhibitory effects of nitric oxide on the expression and activity of aromatase in human granulosa cells. Mol Hum Reprod 5:396–401[Abstract/Free Full Text]
  9. Nishida N, Hattori MA, Takesue K, Kato Y, Fujihara N 2000 Critical role of nitric oxide in expression of porcine LH receptor at transcription and post-transcription levels. Exp Clin Endocrinol Diabetes 108:424–429[CrossRef][Medline]
  10. Matsumi H, Koji T, Yano T, Yano N, Tsutsumi O, Momoeda M, Osuga Y, Taketani Y 1998 Evidence for an inverse relationship between apoptosis and inducible nitric oxide synthase expression in rat granulosa cells: a possible role of nitric oxide in ovarian follicle atresia. Endocr J 45:745–751[Medline]
  11. Jablonka-Shariff A, Olson LM 1998 The role of nitric oxide in oocyte meiotic maturation and ovulation: meiotic abnormalities of endothelial nitric oxide synthase knock-out mouse oocytes. Endocrinology 139:2944–2954[Abstract/Free Full Text]
  12. Tamura N, Doolittle LK, Hammer RE, Shelton JM, Richardson JA, Garbers DL 2004 Critical roles of the guanylyl cyclase B receptor in endochondral ossification and development of female reproductive organs. Proc Natl Acad Sci USA 101:17300–17305[Abstract/Free Full Text]
  13. Tornell J, Carlsson B, Billig H 1990 Atrial natriuretic peptide inhibits spontaneous rat oocyte maturation. Endocrinology 126:1504–1508[Abstract/Free Full Text]
  14. Zhang M, Tao Y, Zhou B, Xie H, Wang F, Lei L, Huo L, Sun Q, Xia G 2005 Atrial natriuretic peptide inhibits the actions of FSH and forskolin in meiotic maturation of pig oocytes via different signalling pathways. J Mol Endocrinol 34:459–472[Abstract/Free Full Text]
  15. Conti M 2002 Specificity of the cyclic adenosine 3',5'-monophosphate signal in granulosa cell function. Biol Reprod 67:1653–1661[Abstract/Free Full Text]
  16. Rentero C, Monfort A, Puigdomenech P 2003 Identification and distribution of different mRNA variants produced by differential splicing in the human phosphodiesterase 9A gene. Biochem Biophys Res Commun 301:686–692[CrossRef][Medline]
  17. Laforest MF, Pouliot E, Gueguen L, Richard FJ 2005 Fundamental significance of specific phosphodiesterases in the control of spontaneous meiotic resumption in porcine oocytes. Mol Reprod Dev 70:361–372[CrossRef][Medline]
  18. Francis SH, Sekhar KR, Rouse AB, Grimes KA, Corbin JD 2003 Single step isolation of sildenafil from commercially available Viagra tablets. Int J Impot Res 15:369–372[CrossRef][Medline]
  19. Campagna C, Ayotte P, Sirard MA, Arsenault G, Laforest JP, Bailey JL 2007 Effect of an environmentally relevant metabolized organochlorine mixture on porcine cumulus-oocyte complexes. Reprod Toxicol 23:145–152[CrossRef][Medline]
  20. Sasseville M, Cote N, Guillemette C, Richard FJ 2006 New insight into the role of phosphodiesterase 3A in porcine oocyte maturation. BMC Dev Biol 6:47[CrossRef][Medline]
  21. Petters RM, Wells KD 1993 Culture of pig embryos. J Reprod Fertil Suppl 48:61–73[Medline]
  22. Beavo JA, Hardman JG, Sutherland EW 1970 Hydrolysis of cyclic guanosine and adenosine 3',5'-monophosphates by rat and bovine tissues. J Biol Chem 245:5649–5655[Abstract/Free Full Text]
  23. Thompson WJ, Terasaki WL, Epstein PM, Strada SJ 1979 Assay of cyclic nucleotide phosphodiesterase and resolution of multiple molecular forms of the enzyme. Adv Cyclic Nucleotide Res 10:69–92[Medline]
  24. Brown DA, Rose JK 1992 Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell 68:533–544[CrossRef][Medline]
  25. Bouillon M, El Fakhry Y, Girouard J, Khalil H, Thibodeau J, Mourad W 2003 Lipid raft-dependent and -independent signaling through HLA-DR molecules. J Biol Chem 278:7099–7107[Abstract/Free Full Text]
  26. Harder T, Scheiffele P, Verkade P, Simons K 1998 Lipid domain structure of the plasma membrane revealed by patching of membrane components. J Cell Biol 141:929–942[Abstract/Free Full Text]
  27. Zhang X, Feng Q, Cote RH 2005 Efficacy and selectivity of phosphodiesterase-targeted drugs in inhibiting photoreceptor phosphodiesterase (PDE6) in retinal photoreceptors. Invest Ophthalmol Vis Sci 46:3060–3066[Abstract/Free Full Text]
  28. Lugnier C 2006 Cyclic nucleotide phosphodiesterase (PDE) superfamily: a new target for the development of specific therapeutic agents. Pharmacol Ther 109:366–398[CrossRef][Medline]
  29. Lin CS, Chow S, Lau A, Tu R, Lue TF 2002 Human PDE5A gene encodes three PDE5 isoforms from two alternate promoters. Int J Impot Res 14:15–24[CrossRef][Medline]
  30. Loughney K, Hill TR, Florio VA, Uher L, Rosman GJ, Wolda SL, Jones BA, Howard ML, McAllister-Lucas LM, Sonnenburg WK, Francis SH, Corbin JD, Beavo JA, Ferguson K 1998 Isolation and characterization of cDNAs encoding PDE5A, a human cGMP-binding, cGMP-specific 3',5'-cyclic nucleotide phosphodiesterase. Gene 216:139–147[CrossRef][Medline]
  31. Deterre P, Bigay J, Forquet F, Robert M, Chabre M 1988 cGMP phosphodiesterase of retinal rods is regulated by two inhibitory subunits. Proc Natl Acad Sci USA 85:2424–2428[Abstract/Free Full Text]
  32. Viczian AS, Piriev NI, Farber DB 1995 Isolation and characterization of a cDNA encoding the {alpha}' subunit of human cone cGMP-phosphodiesterase. Gene 166:205–211[CrossRef][Medline]
  33. Cote RH 2007 Photoreceptor phosphodiesterase (PDE6): a G-protein-activated PDE regulating visual excitation in rod and cone photoreceptor cells. In: Beavo JA, Francis SH, Houslay MD, eds. Cyclic nucleotide phosphodiesterases in health and disease. Boca Raton, FL: CRC Press; 713
  34. Gutkowska J, Jankowski M, Sairam MR, Fujio N, Reis AM, Mukaddam-Daher S, Tremblay J 1999 Hormonal regulation of natriuretic peptide system during induced ovarian follicular development in the rat. Biol Reprod 61:162–170[Abstract/Free Full Text]
  35. Noubani A, Farookhi R, Gutkowska J 2000 B-type natriuretic peptide receptor expression and activity are hormonally regulated in rat ovarian cells. Endocrinology 141:551–559[Abstract/Free Full Text]
  36. Sriraman V, Rudd MD, Lohmann SM, Mulders SM, Richards JS 2006 Cyclic guanosine 5'-monophosphate-dependent protein kinase II is induced by luteinizing hormone and progesterone receptor-dependent mechanisms in granulosa cells and cumulus oocyte complexes of ovulating follicles. Mol Endocrinol 20:348–361[Abstract/Free Full Text]
  37. Goff AK, Major PW 1976 Bovine luteal cyclic AMP and cyclic GMP phosphodiesterase activity; sensitivity to various drugs and hormones. FEBS Lett 61:144–147[CrossRef][Medline]
  38. Vollmar AM, Mytzka C, Arendt RM, Schulz R 1988 Atrial natriuretic peptide in bovine corpus luteum. Endocrinology 123:762–767[Abstract/Free Full Text]
  39. Pandey KN, Osteen KG, Inagami T 1987 Specific receptor-mediated stimulation of progesterone secretion and cGMP accumulation by rat atrial natriuretic factor in cultured human granulosa-lutein (G-L) cells. Endocrinology 121:1195–1197[Abstract/Free Full Text]
  40. Budnik LT, Brunswig B, Mukhopadhyay AK 1987 Atrial natriuretic factor stimulates luteal guanylate cyclase. Regul Pept 19:23–34[CrossRef][Medline]
  41. LaPolt PS, Hong LS 1995 Inhibitory effects of superoxide dismutase and cyclic guanosine 3',5'-monophosphate on estrogen production in cultured rat granulosa cells. Endocrinology 136:5533–5539[Abstract]
  42. Johnson MC, Diaz HA, Stocco C, Palomino A, Devoto L, Vega M 1999 Antisteroidogenic action of nitric oxide on human corpus luteum in vitro: mechanism of action. Endocrine 11:31–36[CrossRef][Medline]
  43. Sasseville M, Cote N, Vigneault C, Guillemette C, Richard FJ 2007 3'5'-Cyclic adenosine monophosphate-dependent up-regulation of phosphodiesterase type 3A in porcine cumulus cells. Endocrinology 148:1858–1867[Abstract/Free Full Text]
  44. Faerge I, Terry B, Kalous J, Wahl P, Lessl M, Ottesen JL, Hyttel P, Grondahl C 2001 Resumption of meiosis induced by meiosis-activating sterol has a different signal transduction pathway than spontaneous resumption of meiosis in denuded mouse oocytes cultured in vitro. Biol Reprod 65:1751–1758[Abstract/Free Full Text]
  45. Richard FJ, Tsafriri A, Conti M 2001 Role of phosphodiesterase type 3A in rat oocyte maturation. Biol Reprod 65:1444–1451[Abstract/Free Full Text]
  46. Bender AT, Beavo JA 2006 Cyclic nucleotide phosphodiesterases: molecular regulation to clinical use. Pharmacol Rev 58:488–520[Abstract/Free Full Text]
  47. Liu H, Seno K, Hayashi F 2003 Active transducin {alpha} subunit carries PDE6 to detergent-resistant membranes in rod photoreceptor outer segments. Biochem Biophys Res Commun 303:19–23[CrossRef][Medline]
  48. Florio SK, Prusti RK, Beavo JA 1996 Solubilization of membrane-bound rod phosphodiesterase by the rod phosphodiesterase recombinant {delta} subunit. J Biol Chem 271:24036–24047[Abstract/Free Full Text]
  49. Ma L, Wang HY 2007 Mitogen-activated protein kinase p38 regulates the Wnt/cyclic GMP/Ca2+ non-canonical pathway. J Biol Chem 282:28980–28990[Abstract/Free Full Text]
  50. Hernandez-Gonzalez I, Gonzalez-Robayna I, Shimada M, Wayne CM, Ochsner SA, White L, Richards JS 2006 Gene expression profiles of cumulus cell oocyte complexes during ovulation reveal cumulus cells express neuronal and immune-related genes: does this expand their role in the ovulation process? Mol Endocrinol 20:1300–1321[Abstract/Free Full Text]
  51. Villa-Diaz LG, Miyano T 2004 Activation of p38 MAPK during porcine oocyte maturation. Biol Reprod 71:691–696[Abstract/Free Full Text]
  52. Park JY, Richard F, Chun SY, Park JH, Law E, Horner K, Jin SL, Conti M 2003 Phosphodiesterase regulation is critical for the differentiation and pattern of gene expression in granulosa cells of the ovarian follicle. Mol Endocrinol 17:1117–1130[Abstract/Free Full Text]
  53. Conti M, Kasson BG, Hsueh AJ 1984 Hormonal regulation of 3',5'-adenosine monophosphate phosphodiesterases in cultured rat granulosa cells. Endocrinology 114:2361–2368[Abstract/Free Full Text]
  54. Michael AE, Webley GE 1991 Prostaglandin F2 {alpha} stimulates cAMP phosphodiesterase via protein kinase C in cultured human granulosa cells. Mol Cell Endocrinol 82:207–214[CrossRef][Medline]
  55. Sasson R, Rimon E, Dantes A, Cohen T, Shinder V, Land-Bracha A, Amsterdam A 2004 Gonadotrophin-induced gene regulation in human granulosa cells obtained from IVF patients. Modulation of steroidogenic genes, cytoskeletal genes and genes coding for apoptotic signalling and protein kinases. Mol Hum Reprod 10:299–311[Abstract/Free Full Text]



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